Malignant hyperthermia (MH) is a pharmacogenetic disease of skeletal muscle. Our long term objectives are to understand how mutations of RyR1and the a[lpha]1DHPR that are associated with MH alter the process of excitation contraction coupling, and to define if different MH mutations have different phenotypes and/or influence the phenotypic penetrance of human MH. This is a competitive supplemental application. Hypothesis: Mutations in RyR1 and the a1DHPR associated with human MH influence the ability of RyR1, the a1DHPR, and FKBP12 to interact with and regulate RyR1 function. These changes lead to a common pattern of dysfunction of antegrade and retrograde signaling among these proteins that triggers MH. The specific aims of the funded application were: 1). To create and express, mutated cDNAs with RyR1 MH mutations Arg163Cys, Gly341Arg, Arg614Cys, Va12168Met, Arg2163Cys, and Arg2458His and the a1DHPR mutation, Argl086His, in null skeletal myotubes and elucidate the effect of these mutations on macroscopic Ca2+ fluxes, [3H]ryanodine binding, single-channel gating kinetics and the structural integrity of the FKBP12-RyR1 complex. 2). To create four homozygous transgenic mouse lines (RyR1 Gly341Arg, Arg614Cys, Arg2458His and alDHPR Argl086His), each expressing one of these mutated proteins and measure their responses to halogenated volatile anesthetics and depolarizing neuromuscular blockers in vivo, and their responses to clinical in vitro contracture tests (IVCT). Additional Supplement Aims: We have found that there are broad phenotypic differences among RyRl MH mutations in responsiveness to K+ depolarization, and the direct agonists caffeine and 4-chloro-rn-cresol. Others have recently shown that dysfunction of EC coupling caused by CCD mutations are directly related to intracellular Ca2+ concentration ([Ca2+]i). We propose 1) To directly measure [Ca2+Ii in our MH cells with Ca2+ selective microelectrodes. 2.) To define the effect of non-cell permeant agents such as IP3, cADPR and ruthenium red on [Ca2+]i in MH myotubes. Because data in our progress report show that we can not create MH mice using the "rescue transgenic" approach, 3) We are forced to turn to our previously proposed but more expensive alternative using homologous recombination to create "knock in" mice with MH mutations.